Protein kinase C, PKC, is a family of widely distributed signal transduction proteins important for cell growth, differentiation, and other responses. PKC is activated by growth factors, hormones, and other external messengers via stimulation of phospholipase C and the generation of the second messengers, inositol triphosphate and diacylglycerol. All members of the PKC family share significant sequence homology and perform signal transduction via protein phosphorylation. Almost all cell types express one or more isoforms of PKC.
The activity of an endogenous inhibitor of PKC has been detected in bovine, avian, murine, and human tissues. The first complete primary structure of an endogenous inhibitor of protein kinase C, PKCI-1, was derived from bovine brain (Pearson, J D et al (1990) J Biol Chem 265: 4583-4591). PKCI-1 has a specific site of interaction with PKC and inhibits the phosphorylation ability of PKC. In addition to the bovine sequence, a complete amino acid sequence of PKCI-1 has been deduced from the maize, rat, and human genes (Simpson G G et al (1994) Biochim Biophys Acta 1222: 306-308; Waller S J and Murphy D (1994), unpublished; Brzoska P M et al (1995) PNAS USA 92: 7824-7828). The bovine PKCI-1 was shown to be a zinc (Zn) binding protein (Simpson et al, supra). The site of Zn binding has been localized to a 11 amino acid fragment (Mozier et al (1991) FEBS Lett 279:14-18), and the Zn binding domain is conserved among PKCI-1 molecules of other species.
Disease and PKC
PKC activation plays a significant role in multidrug resistance (MDR), a major contributing factor in the failure of many chemotherapeutic cancer regimens (Grunicke H et al (1994) Ann Hematol 69: S1-6). PKC regulates proteins that act to make cancer cells drug resistant, such as FOS, Jun, glutathione S-transferase, deoxy-thymidine monophosphate synthase, metallothionein, and mdr-1-encoded P-glycoprotein. PKC leads to the increase in transcription of these genes through protein phosphorylation. PKC inhibitors or antagonists that interfere with the phosphorylation function of PKC have been shown to reduce the expression of genes that mediate MDR. Evidence suggests that the selective inhibition of a particular PKC, cPKC alpha, might allow successful management of colon cancer with standard cytotoxic chemotherapeutic regimens (O'Brian C A et al (1995) Growth Factors and Tumor Promotion: Implication for Risk Assessment, Wiley-Lis, Inc., pages 117-120). Thus, PKC is a promising agent for cancer treatment.
In addition to contributing to MDR, PKC activation is often a critical event in tumor promotion (O'Brian C A and Ward N E (1989) Cancer Metastasis Rev 8: 199-214). For example, PKC alpha phosphorylates and activates the growth promoting gene Raf-1 (Kolch W et al (1993) Nature 364: 249-252). Without PKC induced Raf-1 activation, the ability to transform NIH3T3 cells is greatly inhibited. In cultured melanocytes loss of a particular PKC, isotype has been implicated in cellular transformation (Yamanishi D T et al (1994) Crit Rev Oncog 5: 429-450).
PKC participates in the sequence of molecular events that underlie learning and memory (Olds J L and Alkon D L (1991) New Biol 3: 27-35). The cellular distribution of PKCs changes as a result of memory storage in cells that have been demonstrated to act in memory and learning (Saito N et al (1994) Brain Res 656: 245-256). Thus, molecules that modulate PKC may modify the ability to learn and remember. PKC gamma mutant mice exhibit mild deficits in spatial and contextual learning, further implicating PKCs in learning and memory (Abeliovich A et al (1993) Cell 75: 1263-1271). Memory deterioration is one of the main characteristics and earliest signs of Alzheimer's disease.
PKCs can act in several ways to stimulate apoptosis (programmed cell death) in various cell types. PKCs can block the activation of other calcium-dependent enzymes triggering apoptosis (Lucas M et al (1995) Gen Pharmacol 26: 881-887). Activation of phosphatases by ceramide and inhibition of PKC by sphingosine mediates the sphingomyelin pathway to apoptosis. A putative PKC target, p34cdc2, can act to stimulate apoptosis when its activity is uncoupled from the completion of DNA replication. In addition, p21Ras mediates proliferative responses and also renders cells susceptible to apoptosis after inhibition of PKC activity (Chen C Y et al (1996) J Biol Chem 271: 2376-2379). The T lymphocytes of mice in which the Fas mediated apoptosis pathway has been knocked out rely on a PKC dependent apoptotic mechanism for selection against self-reactive T-cells (Ohkusu K et al (1995) Eur J Immunol 25: 3180-3186). Thus, PKC has a role in immune cell development. Saikosponin b2 induces apoptosis in B16 melanoma cells by down regulation of PKC activity (Zong et al (1996) Biochem Biophys Res Commun 219: 480-485). Furthermore, proteolytic activation of PKC delta by an ICE-like protease stimulates apoptosis in human tumor cell line U-937 (Emoto Y et al (1995) EMBO J 14: 6148-5156).
Many synthetic inhibitors of PKC have been reported, such as chlorpromazine (Mori T et al (1980) J Biol Chem 255: 8378-8380), trifluoperazine (Schatzman R C et al (1981) Biophys Res Commun 98: 669-676), tamoxifen (O'Brien C A et al (1985) Cancer Res 45:
2462-2465), aminoacridines (Hannun Y A et al (1988) J Biol Chem 263: 5124-5131), isoquinolinesulfonamides (Hidaka H et al (1984) Biochemistry 23: 5036-5141), and various synthetic peptides (House C and Kemp B E (1987) Science 238: 1726-1728; Ward N E et al (1995) Cancer Lett 88: 37-40). There is much evidence to suggest that PKC blockers may modify PKC activity in normal or disease cells. For example, PKC blockers have been shown to inhibit the growth of cancer cells both in vitro and in vivo (Levitzki A (1994) Eur J Biochem 226: 1-13).
The PKC inhibitor bisindolylmaleimide GF109203X (bisi) can affect the neuroblastoma cell line Neuro-2A in one of two ways. Without serum, neurite outgrowth is potentiated by bisi, while with serum, apoptosis occurs (Behrens MM et al Cell Growth Differ (1995) 6: 1375-1380). Other PKC inhibitors, including hypericin, staurosporine, tamoxifen, and the phorbol ester PMA induce apoptosis in the human neuroblastoma cell line SK-N-SH (Zhang W et al (1995) Cancer Lett 96: 31-35). PKC blockers may have utility beyond cancer indications. The PKC inhibitor H-7 induces apoptosis in Fas minus mouse T lymphocytes (Ohkusu et al (1995) Eur J Immunol 25: 3180-3186). It is possible that PKC inhibitors may help manage auto-immune diseases by inducing apoptosis in self-reactive T cells. However, synthetically made PKC inhibitors have often failed to advance in drug development because of a high level of toxicity and/or a low level of specificity. The selective inhibition of PKC may allow successful management of diseases associated with PKC induced effects, such as cancer, memory disorders, and auto-immune diseases. Therefore the identification of novel PKC inhibitors provides opportunities for early treatment of diseases associated with PKC.